1. Field of the Invention
The field of the present invention concerns the disinfection of biological materials and in particular concerns a novel use of an insolubilized organic detergent as a disinfecting agent.
2. Description of Related Art
The present inventor has long been concerned with problems of blood borne infection. In particular, he has dealt with methods for disinfecting clotting factors and other proteins purified from blood. He has invented methods of disinfection involving iodine (see, for example, U.S. Pat. Nos. 5,360,605, and 5,370,869), and glycyrrhic triterpenoid and its derivatives (see, for example, U.S. Pat. No. 5,204,324). In addition, he was the inventor of a method of plasma protein purification employing xe2x80x9camphiphilesxe2x80x9d which are more commonly known as detergents (see U.S. Pat. No. 4,314,997).
Literally amphiphile means a substance that has affinities xe2x80x9con both sides.xe2x80x9d This refers to a material that is both hydrophilic (dissolves readily in water) and hydrophobic (dissolves readily in organics such as lipids). Because of its dual nature, such a material can be used to dissolve or emulsify fatty organic substances into an aqueous solution as in removing dirt from clothing. Generally, a detergent is a molecule that is lipophilic (hydrophobic) at one end and hydrophilic at the other end. The hydrophilic end may be hydrophilic by virtue of charged groups, either negative (anionic) or positive (cationic) or may be hydrophilic by virtue of polar but uncharged (nonionic) groups such as hydroxyl groups or oxygen atoms.
This dual hydrophobic/hydrophilic nature gives detergents or amphiphiles favorable properties in purification of therapeutic blood proteins although they may also denature proteins. Blood proteins can be contaminated with any of a number of disease organisms including viruses causing AIDS and hepatitis. Many important disease-causing viruses are composed of a nucleic acid core surrounded by a lipid membrane. It has been shown that detergents can be effective at inactivating viruses. It seems likely that this is due to the detergent emulsifying or otherwise disrupting lipid structures essential for viral activity. One real problem with detergents is that they are also capable of disrupting other vital lipid-based structures like the biomembranes that surrounds and form a significant internal structural component of every animal and plant cell. It turns out to be difficult to find detergents that are sufficiently active to disrupt infective agents while being gentle enough to spare living cells. As a result, huge numbers of detergent structures have been screened looking for optimal detergents. U.S. Pat. No. 4,314,997, mentioned above, gives a lengthy list of candidate detergents.
At the risk of simplifying a hugely complex area it can be considered that ionic detergents (either anionic or cationic) are the most active, and while being very effective at destroying viruses, may readily destroy or damage living cells. Generally, one way to overcome cell destruction is to lower the working concentration of the detergent. However, it is often the case that when the detergent concentration is sufficiently lowered to avoid cell damage, it is also too low to destroy viruses.
The nonionic (uncharged) detergents are generally less active at destroying or damaging cells. Hence it may be possible to find concentrations of nonionic detergents that destroy virus without excessively damaging cells. However, because these detergents are relatively less active, rather high concentrations of detergent are required to adequately destroy viruses. For example, U.S. Pat. No. 4,314,997 claims the broad concentration range of 0.25% to 10% by weight of a variety of detergents. However, the preferred concentration of one nonionic detergent, Triton X-100 (t-octylphenoxypolyethoxyethanol), is at least 2%, while other procedures may use even higher concentrations of detergent. It should be apparent that this may be a case of trading one difficulty for another. Triton X-100, like most detergents, is extremely harmful when injected intravenously. Therefore, removing detergents after they are used for disinfection becomes a very real and significant problem. This problem is merely exacerbated where an extremely high concentration of detergent is used, and especially when one considers that micellar characteristics make it difficult to remove Triton X-100 by dialysis.
One popular method of disinfecting blood products is the so-called xe2x80x9csolvent detergentxe2x80x9d process. In this process plasma viruses are inactivated by the addition of relatively high concentrations of detergents together with an organic solvent-tri-n-butyl-phosphate. The detergent and solvent are then removed by partitioning the protein solution against an organic liquid. The detergent and solvent partition into this liquid and are, hence, eliminated. Most often bland organic liquids such as castor or soy bean oil are used. The oil is then removed by hydrophobic chromatography. It is not difficult to imagine the time and expense of partitioning the plasma and of regenerating or replacing the chromatographic components. Therefore, there remains a significant need for a disinfecting method with the advantages of detergents but which the usual problems of detergent removal.
It is an object of the present invention to provide an improved detergent-based method for inactivation of disease-causing organisms;
It is a further object of the present invention to provide an inactivation method which uses detergent in an easily removable form;
It is an additional object of the present invention to provide a novel disinfecting reagent in the form of a complex between a detergent and a detergent-binding material.
These and other objects are met in an inactivation method that employs a detergent such as nonionic, cationic or anionic detergents and preferably a xe2x80x9csugar detergentxe2x80x9d such as octyl-glucopyranoside. This detergent is effective at inactivating pathogens even when bound by a solid support. Under these conditions the concentration of detergent free in solution is vanishingly low: probably well below one millimolar in concentration. Addition of insoluble detergent results in effective destruction of enveloped viruses in a variety of protein-containing solutions such as clotting factors or other proteins purified from human blood. Because the detergent is essentially entirely bound to a solid substrate, there is little or no difficulty in ensuring that the end product is detergent-free. Because the detergent is so bound, it causes essentially no damage to proteins, blood cells and other cellular material.